木黴菌Trichoderma harzianum ETS323植酸酶之
基因選殖、表達、及基因特性分析

植酸酶(Phytase)是一類可以水解植酸(Phytic acid)生成肌醇與磷酸的酵素。文獻報導指出:於自然界中有的微生物能分泌植酸酶分解難溶性植酸鹽並釋出磷酸，進而促使個體與植物生長。木黴菌Trichoderma harzianum為廣泛應用於生物防治的真菌之一，而其植酸酶的相關研究甚少。本論文研究成功地利用RT-PCR及5ˊRACE等技術由T. harzianum ETS323選殖出植酸酶的gene 和cDNA全長片段(Genomic-phytase-full 和phytase-full)，其核苷酸長度分別為1918 及1680 bp。其中，經分析後發現Genomic-phytase-full 含5段exons 與 4段introns 。演譯後的Phytase經預測發現具有N端訊號胜肽，其成熟蛋白質的分子量為58.4 kDa，其pI值為5.3，並且具有10個可能的醣基化位以及 Histidine-acid phosphatases的高度保留區之胺基酸序列(RHGARYPT)。於大腸桿菌中以胞涵體形式表達出Phytase的部分蛋白 (即Phytase-m)，亦完成了 Phytase-M抗體之製備。雖然於大腸桿菌中以水溶性蛋白形式表達出約全長的Phytase ，但是未偵測出此重組蛋白的大腸桿菌具有Phytase活性。此外，ETS323 Phytase無法在酵母菌Pichia分泌表達。將T. harzianum ETS323 培養於液態PSM (phytase-screening medium)。西方墨點法分析發現Phytase-M抗體可以辨識到 ETS323菌絲體及培養液的蛋白質條帶。然而MALDI-TOF分析顯示這些蛋白質條帶不是Phytase 。而菌絲體及培養液皆未有明顯的Phytase 活性。RT-PCR顯示ETS323 Phytase基因在液固態培養條件中會受到Phytate誘導表現。綜合研究結果推測只有植酸為其磷來源時ETS323無法有效大量誘導分泌產生植酸酶，但仍然可以分泌少許的植酸酶。

關鍵字

Trichoderma harzianum ETS323 植酸酶RT-PCR ； 5ˊRACE ；植酸酶篩選性培養基(PSM) ；誘導表達

並列摘要

The soil borne micro-organisms may secrete phytase to decompose insoluble phytate and release phosphate, subsequently promote plant growth. There is limited study with phytase from Trichoderma harzianum, a well know biological control against plant pathogens. In this study, the full-length of DNA fragment (phytase-full) encoding T. harzianum ETS323 phytase was obtained by RT-PCR and 5ˊ-RACE, as well as ETS323 phytase gene by PCR (Genomic-phytase-full ). The 1918 bp DNA fragment of Genomic-phytase-full is composed of five exons, separated by four introns. phytase-full comprises 1680 bp and its predicted protein has N-terminal signal peptide . The molecular mass of predicted mature ETS323 phytase is 58.4 kDa with 5.3 of pI. A high conserved regin (RHGARYPT) is found in ETS323 phytase, which is conserved among histidine acid phosphatases. ETS323 phytase is probably a glycoprotein as there are ten putative N-glycosylation sites. ETS323 phytase was expressed as soluble proteins in Escherichia coli; however, no phytase activity was detected. ETS323 phytase was not successfully expressed in yeast Pichia. The crude proteins were prepared from T. harzianum ETS323 liquid-cultured in phyate containing medium. Western blot analyses showed that the antibodies raised against EST323 phytase could recognize a protein bands from this culture medium. Moreover, Quantitive RT-PCR analyses indicated that the transcript level of ETS323 phytase could be induced by phytate.